Abstract
Polysialic acid (polySia) is a highly anionic polymer of sialic acid that mostly modifies the neural cell adhesion molecule (NCAM), and involved in brain functions such as learning, memory, circadian rhythm, and social behaviors, through making repulsive and attractive fields on the cell surface. Recently, polySia has been reported to have relationships with some diseases such as psychiatric disorders and cancers. To understand functions of polySia and to apply evaluation methods of polySia structures to the diagnosis of diseases, the comparison of the methods for determining the quality and quantity of polySia structures would be important and necessary. In this study, two monoclonal antibodies, 12E3 and 735, of distinct binding properties were chosen for immunochemical methods by an enzyme-linked immunosorbent assay (ELISA) and a Western-blotting. Two chemical methods, mild acid hydrolysis-fluorometric anion exchange chromatography (MH-FAEC) analysis, and fluorometric C7/C9 analysis were also chosen. These methods were applied to a small amount of crude adult and embryonic brain homogenates to evaluate the quantity and quality of polySia structure. The quantity of polySia can be evaluated using anti-polySia antibodies by ELISA. In addition, MH-FAEC can be also applied to the evaluation of both quantity and quality (chain length) of polySia chain. Biochemical characters of polySia-NCAM in crude homogenates can be analyzed using a mono Q-anion exchange chromatography. Combinational analyses of these methods could help our further understanding of polySia structure, and the quantity and quality of polySia will become criteria useful for diagnosis of diseases.
Highlights
Polysialic acid is a polymer of sialic acid with a degree of polymerization (DP) ranging from 8 to 400 and is mainly attached to neural cell adhesion molecule (NCAM) in brains of vertebrates [1]
The proportion of the immunoreactivity against pig adult brain (PAB) to pig embryonic brain (PEB) was 21% and 22% for 12E3 and 735, respectively (Figure 1, 12E3 and 735, Table 1). These results indicate that the quantity of Polysialic acid (polySia) in PAB is reduced from that in PEB by 80%
With 735 antibody, the amount of polySia determined by Western-blotting was two times lower than that determined by enzyme-linked immunosorbent assay (ELISA) (Table 1). These results indicate that polySia-NCAM species that are detectable with 735 antibody in ELISA might not be effectively immobilized on Polyvinylidene difluoride (PVDF) membrane probably because of longer polySia chain attached on the NCAM
Summary
Polysialic acid (polySia, PSA) is a polymer of sialic acid with a degree of polymerization (DP) ranging from 8 to 400 and is mainly attached to neural cell adhesion molecule (NCAM) in brains of vertebrates [1]. To understand the polySia function, several types of polySia-related enzyme deficient mice were developed. ST8SIA2 and ST8SIA4 double KO (DKO) mice have no polySia structure using mAb. 735 as a polySia-detecting probe and show a severe phenotype, characterized by postnatal growth retardation, precocious death, high incidence of hydrocephalus and agenesis, and hypoplasia of major brain fiber tract [7]. Because almost all DKO mice die soon after birth (80% die before the age of 4 weeks), it appears that the presence of polySia plays a direct and important role in brain functions. ST8SIA2 single KO (SKO) [8] and ST8SIA4 SKO mice [9]
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